We have characterized inhibition of the aspartic protease endothiapepsin by several transition-state analogues (H-142, H-77, H-189 and H-261). Time-dependent competitive inhibition was observed in all cases that can be explained in terms of the rapid formation of a transient-state and its subsequent slow isomerization to form the final complex. Significant differences in the inhibition constants were observed (from 0.07 nM for H-261 to 1.9 nM for H-77). The binding process was shown to be both enthalpically and entropically favoured at room temperature. The structural parameterization of the binding energetics recently developed in this laboratory was applied to this system. It was shown that the differences in inhibitory capability of these inhibitors is due to the differences in the ionization process coupled to the binding event. In fact, there is a linear correlation between the number of protons released upon binding and the apparent free energy change upon binding. The estimated intrinsic inhibition constant (2 pM) is in agreement with the value predicted from the structural changes.